Repeated stress exposure produces a dysphoria-like response in mice that manifests behaviorally as stress-induced immobility, aversion and social avoidance. In a series of recent studies, we found that exposure to different forms of sustained stress activates a cascade of neurochemical responses in brain involving CRF activation of dynorphin release, dynorphin activation of kappa opioid receptors, kappa stimulation of p38alpha MAPK, and subsequent translocation of the serotonin transporter (SERT) from an endosomal compartment to the plasma membrane. Conditional gene deletion approaches and neurochemical studies revealed that the stress-induced aversion response specifically required the dynorphin/Kappa/p38alpha MAPK/SERT in the dorsal raphe serotonergic nerve terminals innervating the ventral striatum; disruption of any one of these components genetically or pharmacologically blocked the stress-induced dysphoric response and conferred stress-resilience in these animal models. Because stress-vulnerability is a known risk factor for clinical depression in humans, the proposed studies are designed to further characterize the mechanisms responsible for SERT translocation in the serotonergic nerve terminals. Specifically, we propose to express the human-SERT gene by local injection of a lentiviral construct containing the normal hSERT coding sequence into dorsal raphe neurons of SERT(-/-) mice. We previously showed that this restores SERT functionality and stress-vulnerability in the mice. In aim 1, we would alter the hSERT coding region in the lenti-hSERT by a systematic site-directed mutagenesis approach to define the structural features of SERT necessary for stress-induced translocation. Natural variants of hSERT have been identified in the human population by genome sequencing, and some of these variants have been described as conferring disease risk to the affected individuals. In aim 2, we would engineer lenti-hSERT to express these natural variants, then determine the effects of the resulting structural changes on the stress-response in mice. SERT(-/-) mice expressing different forms of the lenti-hSERT would be assessed 1) behaviorally for stress-induced aversion responses, 2) neurochemically for stress-induced translocation of SERT protein to plasma membrane of synaptosomes isolated from ventral striata, and 3) biophysically using rotating disk electrovoltammetry to define the effects f stress on serotonin transport kinetics. The proposed studies would better define how stress-induced changes in serotonin transport in the ventral striatum might control the risk of depression-like behaviors in mice, and the proposed analysis of the natural human variants of SERT might help describe a possible genetic basis for individual differences in stress-resilience.